四种食源性致病弧菌多重实时荧光定量PCR快速检测体系的建立

A multiplex real time fluorescence quantitative PCR method for rapid detection of four foodborne pathogenic species of Vibrio

摘要:

【背景】弧菌在全球范围内严重威胁人类健康，副溶血性弧菌(Vibrio parahaemolyticus)、霍乱弧菌(Vibrio cholerae)、拟态弧菌(Vibrio mimicus)和创伤弧菌(Vibrio vulnificus)与食用生的或未煮熟的海鲜所引起的胃肠道感染和败血症有关，因而备受关注。【目的】建立一种实时荧光定量PCR方法，用于检测副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌，以提高检测效率和准确性。【方法】基于副溶血性弧菌和拟态弧菌的toxR基因、霍乱弧菌的ompW基因、创伤弧菌的vvhA基因设计特异性引物和探针，通过优化反应体系和条件建立四重实时荧光定量PCR体系。【结果】该实时荧光定量PCR方法检测限均为10 copies/µL，扩增效率均在100%左右；在特异性试验中，分别运用多重实时荧光定量PCR和常规PCR对目的菌基因组、非目的菌基因组和空白对照进行扩增，结果均只有目的弧菌扩增明显，表明本方法有良好的特异性；在抗干扰实验中，高浓度弧菌不干扰低浓度弧菌检测；每个浓度梯度进行3次重复实验，每组变异系数均小于1.5%，表明本方法重复性好。【结论】建立的多重实时荧光定量PCR方法可快速、特异地实现对副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌的检测。

Abstract:

[Background] Vibrio species pose a serious threat to human health worldwide. Vibrio parahaemolyticus, V.cholerae, V.mimicus and V.vulnificus capable of causing gastrointestinal infections and sepsis associated with consumption of raw or undercooked seafood are of particular concern. [Objective] To develop a real time fluorescence quantitative PCR method with improved efficiency and accuracy for the detection of V.parahemolyticus, V.cholerae, V.mimicus, and V.vulnificus. [Methods] The specific primers and probes were designed based on toxR of V.parahaemolyticus and V.mimicus, ompW of V.cholerae, and vvhA of V.vulnificus. A quadruplex real time fluorescence quantitative PCR method was established by optimizing the reaction system and conditions. [Results] The minimum detection limit of the real time fluorescence quantitative PCR method was 10 copies/µL, and the amplification efficiency was about 100%. In the specificity test, multiplex real time fluorescence quantitative PCR and conventional PCR were used to amplify the target bacteria genome, non-target bacteria genome and blank control strain genome, respectively. The results showed that only the target vibrio genome was amplified significantly, indicating that the method had good specificity. In the anti-interference experiment, high concentration Vibrio did not interfere with the detection of low concentration Vibrio. Three repeated experiments were performed for each concentration gradient, and the coefficient of variation of each group was less than 1.5%, indicating that the method was reproducible in this experiment. [Conclusion] The multiplex real time fluorescence quantitative PCR method was established, which could detect V. parahemolyticus, V. cholerae, V. mimicus, and V. vulnificus specifically and rapidly.